Hyaluronic acid, one of the extracellular matrices, is an important factor related to chemotaxis, wound healing, tumor metastasis and the like, and exists in almost all cells of the living tissues. The intercellular communication mediated by the hyaluronic acid and hyaluronic acid binding receptors on the cell surface is one of the important means of the intercellular interaction. Recently, the hyaluronic acid also has been reported to have ability as an activating factor of NF (nuclear factor)-κB, one of the intranuclear transcription factor, and as an angiogenesis promoting factor, as well as a chemotaxis factor.
Several hyaluronic acid binding receptors located on cell membrane for example, CD44 (one of Clusters of Differentiation, i.e., lymphocyte antigen groups), LYVE-1 (lymphatic vessel endothelial HA receptor), RHAMM (Receptor for Hyaluronan-Mediated Motility), and LEC (Liver Endothelial Cell clearance) receptor have been hitherto identified. Among these hyaluronic acid binding receptors, LYVE-1 has been found to be a receptor comprising 322. amino acid residues (see, J. Cell Biol. 144, 789-801 (1999)).
It has been clarified that mRNA of LYVE-1 is expressed in almost whole body, such as the spleen, the lymph node, the fetal liver, the heart, the lung and the like, and that the LYVE-1 receptors are localized on the endothelium of the lymphatic vessel in those tissues. Therefore, it is considered that the LYVE-1 may be used for identifying the lymphatic vessel which is difficult to be discriminated morphologically from the blood vessel.
For example, in the lymphogenous metastasis of malignant, the processes of metastasis are: after invasion to surrounding tissues, the tumor infiltrates the lymphatic vessel wall destructively and penetrates into the lymphatic vessels, where it is transferred lymphogenously, to the region distant from the primary focus. In these invading processes, the host tissues involving the lymphatic vessel are broken.
Accordingly, if detailed observations of the lymphatic vessels in the pathological tissues are obtained, then they may give us useful histopathology of the destructive and invasive growth of the tumor.
Until now, there have been several reports having observed the image of the tumor invading into lymphatic vessels by infiltrating through the lymphatic vessel wall destructively (see: Takazawa H., An electron-microscopic study on the transplanted part of lymphatic vessels in the experimental formation of lymphatic metastasis (in Japanese), General meeting news of Japan Cancer Association, 30, 296(1971); Araki K., An electron-microscopic study of the initial stage of the cancer invasion and the lymphatic vessel infiltration: an observation on an N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG) induced rat stomach cancer (in Japanese), Okayama Med. J., 91, 659-669 (1979); James N., et al., Growth and metastasis of Lewis lung carcinoma in the footpad of mice, Expl. Cell Biol., 56, 221-228 (1992); Carr I., et al., The fine structure of neoplastic invasion: invasion of liver, skeletal muscle and lymphatic vessels by the Rd/3 tumour, J. Pathol., 118, 91-99 (1976); Paku S., et al., Ultrastructural analysis of experimentally induced invasion in the rat lung by tumour cells metastasizing lymphatically, Anticancer Res., 6, 957-966 (1986)).
These reports, however, are the studies at the cell level making use of electron microscopes. There have been no reports on tissue level having observed scenes of tumor invasion into the lymphatic vessels in a tissue.
On the other hand, various staining methods of tissues or the like for observing pathological images of pathological tissues by staining have been developed. For example, immunohistochemical staining techniques such as an enzyme antibody technique or the like are excellent in the specificity and reproducibility, and the method according to the present invention belongs to this category. However, no methods have hitherto been known that it selects the lymphatic vessels as a staining target and stains it selectively.